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Li P.,Institute of Regenerative Medicine and Biofunction | Ninomiya H.,Tottori University | Kurata Y.,Kanazawa Medical University | Kato M.,Tottori University | And 13 more authors.
Circulation Research | Year: 2011

RATIONALE: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current IKr. It is highly expressed in cardiomyocytes and its mutations cause long QT syndrome type 2. Heat shock protein (Hsp)70 is known to promote maturation of hERG. Hsp70 and heat shock cognate (Hsc70) 70 has been suggested to play a similar function. However, Hsc70 has recently been reported to counteract Hsp70. OBJECTIVE: We investigated whether Hsc70 counteracts Hsp70 in the control of wild-type and mutant hERG stability. METHODS AND RESULTS: Coexpression of Hsp70 with hERG in HEK293 cells suppressed hERG ubiquitination and increased the levels of both immature and mature forms of hERG. Immunocytochemistry revealed increased levels of hERG in the endoplasmic reticulum and on the cell surface. Electrophysiological studies showed increased IKr. All these effects of Hsp70 were abolished by Hsc70 coexpression. Heat shock treatment of HL-1 mouse cardiomyocytes induced endogenous Hsp70, switched mouse ERG associated with Hsc70 to Hsp70, increased IKr, and shortened action potential duration. Channels with disease-causing missense mutations in intracellular domains had a higher binding capacity to Hsc70 than wild-type channels and channels with mutations in the pore region. Knockdown of Hsc70 by small interfering RNA or heat shock prevented degradation of mutant hERG proteins with mutations in intracellular domains. CONCLUSIONS: These results indicate reciprocal control of hERG stability by Hsp70 and Hsc70. Hsc70 is a potential target in the treatment of LQT2 resulting from missense hERG mutations. © 2011 American Heart Association, Inc.


Hashimoto M.,Chiba University | Kobayashi K.,Chiba University | Watanabe M.,Chiba University | Kazuki Y.,Institute of Regenerative Medicine and Biofunction | And 9 more authors.
Journal of Lipid Research | Year: 2013

Here , we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a - knockout ( Cyp3a-/- ) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a-/- mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element- binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a-/- mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a-/- mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a-/- mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7 β -hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a-/- mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a-/- mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo. -Hashimoto, M., K. Kobayashi, M. Watanabe, Y. Kazuki, S. Takehara, A. Inaba, S-i. Nitta, N. Senda, M. Oshimura, and K. Chiba. Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.


Miyazaki S.,Nojima Hospital | Hamada T.,Tottori University | Hirata S.,Hirata Clinic of Internal Medicine | Ohtahara A.,Sanin Rosai Hospital | And 13 more authors.
Clinical and Experimental Hypertension | Year: 2014

Purpose: To examine effects of a long-acting calcium channel blocker (CCB) azelnidipine on uric acid metabolism in hypertensive patients. Methods: Azelnidipine was administered to 72 patients at a daily dose of 8omg or 16omg. In 22 cases out of the 72 patients, a different CCB was switched to azelnidipine. Blood pressure was measured and biochemical parameters of blood and urine were evaluated before and 2-3 months after the administration. Results: Azelnidipine significantly decreased both systolic and diastolic blood pressure and the heart rate. It decreased both serum urate levels and the urinary uric acid to creatinine ratio (Uur/Ucr), but did not affect the uric acid clearance to creatinine clearance ratio (Cur/Ccr). Azelnidipine decreased both Uur/Ucr and Cur/Ccr in patients with Uur/Ucr 0.5 or 0.34, although it did not change these clearance parameters in patients with Uur/Ucr <0.5 or <0.34. Azelnidipine decreased the serum urate levels and Uur/Ucr in hyperuricemic patients with uric acid levels 7.0omg/dL in males and 6.0omg/dL in females. It did not change these parameters in normouricemic patients with serum urate levels <7.0omg/dL in males and <6.0omg/dL in females. Azelnidipine decreased Uur/Ucr and Cur/Ccr in hyperuricemic patients with normal or overexcretion of uric acid, although it did not change these clearance parameters in hyperuricemic patients with uric acid hypoexcretion. Conclusions: Azelnidipine decreased the serum urate acid levels and Uur/Ucr, and this response was most prominent in hyperuricemic patients or patients with normal and overexcretion of uric acid. © 2014 Informa Healthcare USA, Inc.


Yamaguchi S.,Institute of Regenerative Medicine and Biofunction | Niwa R.,Bioscience Technology | Kazuki Y.,Tottori University | Ohbayashi T.,Institute of Regenerative Medicine and Biofunction
Yonago Acta Medica | Year: 2011

Exactly controlled conditional gene expressing systems are crucial for genomic functional research, animal transgenesis and gene therapy. Bacterial artificial chromosomes (BACs) are optimal for harboring long fragments of genomic DNA or large cDNA up to 300 kb in size. Therefore, BACs are available to produce transgenic cells and animals for the functional studies of genes. However, BAC can insert DNA randomly into the host genome, possibly causing unpredicted expression. We previously developed a human artifi{ligature}cial chromosome (HAC) vector from human chromosome 21 using chromosome engineering. The HAC vector has several important characteristics desired for an ideal gene delivery vector, including stable episomal maintenance, and the ability to carry large genomic DNA containing its own regulatory element, thus allowing physiological regulation of the transgene in a manner similar to that of the native chromosome. In this study, we develop a system fusing BAC library and HAC technology together to allow tight control of gene expression This system enables BAC to be cloned into the defined locus on the HAC vector by the Cre/loxP system. In addition, the genome in the BAC is possible to be engineered freely by the BAC recombineering technology. This system is a highly efficient tool for the rapid generation of stringently controlled gene expression system on the HAC vector.


Oshimura M.,Institute of Regenerative Medicine and Biofunction | Oshimura M.,Tottori University | Kazuki Y.,Institute of Regenerative Medicine and Biofunction | Kazuki Y.,Tottori University | And 2 more authors.
Clinical Neurology | Year: 2012

Human artificial chromosomes (HACs) are stable episomal gene vectors that can carry large gene inserts. We have reported complete correction of a genetic deficiency following the transfer of a HAC carrying the genomic dystrophin sequence (DYS-HAC) into induced pluripotent stem (iPS) cells derived from either a Duchenne muscular dystrophy (DMD) model mouse or a human DMD patient. The engineered iPS cells could differentiate in immunodeficient nude mice, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice generated from the engineered cells showed tissue-specific expression of dystrophin. Recently, Giulio's group has isolated and characterized a population of blood vessel-associated stem cells, called mesoangioblasts, that can differentiate into multiple mesoderm cell types, including skeletal muscle. The DYS-HAC was transferred to mesoangioblasts from the DMD-model mouse. Thus, when delivered in the arterial circulation, mesoangioblasts crossed the blood vessel wall and participated in skeletal muscle regeneration, ameliorating signs of muscular dystrophy in the DMD model mice. Most recently, the iPS cells from a DMD patient corrected with the DYS-HAC, were successfully differentiated to mesoangioblasts. Therefore, autologous transfer of genetically corrected iPS cells and muscle progenitor cells will be desirable therapeutic cells because immune suppression would not be required.


Iwai C.,Institute of Regenerative Medicine and Biofunction | Li P.,Institute of Regenerative Medicine and Biofunction | Kurata Y.,Kanazawa Medical University | Hoshikawa Y.,Institute of Regenerative Medicine and Biofunction | And 16 more authors.
Cardiovascular Research | Year: 2013

Aims: We examined the role of Hsp90 in expression and maturation ofwild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. Methods and results: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol sup-pressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp 90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquiti-nated form, increased its immunoreactivityin the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Conclusion: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins. © The Author 2013.


Kazuki Y.,Institute of Regenerative Medicine and Biofunction | Kazuki Y.,Tottori University | Kobayashi K.,Chiba University | Aueviriyavit S.,Chiba University | And 19 more authors.
Human Molecular Genetics | Year: 2013

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes. © The Author 2012. Published by Oxford University Press. All rights reserved.


Otsuka S.,Tottori University | Otsuka S.,Institute of Regenerative Medicine and Biofunction | Sakamoto Y.,Tottori University | Siomi H.,Keio University | And 6 more authors.
Brain and Development | Year: 2010

Fragile X syndrome (FXS), which is the most common form of familial mental retardation, is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. Previous studies have suggested that as compared to other populations, Japanese have a lower prevalence of FXS. In addition, in the normal population, there are no carriers who have the premutation allele. We analyzed a total of 946 normal Japanese (576 males and 370 females) and attempted to estimate the frequency of the FMR1 allele. Within this population, we found that 1,155 alleles were in the normal range (less than 40 CGG repeats) and had a modal number of 27 repeats (35.75%). No carriers with premutations (55-200 CGG repeats) were observed in this normal population. We also identified six intermediate-sized alleles (40-54 CGG repeats), with a reported incidence of 1 in 103 males and 1 in 324 females. However, this allele frequency was different from that previously reported for the Japanese population. Since data from previous studies has suggested that FXS might possibly be associated with the genetic mechanism of autism, we also analyzed the length of the CGG repeats in 109 autistic patients. In all cases the CGG repeat numbers were within the normal range (16-36 repeats) and no individuals presented with expanded premutation or intermediate alleles. This finding indicates that the length of the CGG repeat within the FMR1 is unlikely to be responsible for autism in Japanese. © 2008 Elsevier B.V. All rights reserved.


PubMed | Institute of Regenerative Medicine and Biofunction and Tottori University
Type: Journal Article | Journal: European journal of dermatology : EJD | Year: 2014

The pituitary homeobox 1 (PITX1) protein is a member of the bicoid-related homeobox transcription factors and has essential roles in human development. Recently, the PITX1 gene has been considered as a tumor suppressor gene in various human cancers.This study examined the expression of PITX1 in the development and progression of human cutaneous malignant melanoma.Immunohistochemical and/or immunofluorescence analyses were performed to examine the histological expression of PITX1 in healthy skin and 40 cutaneous malignant melanoma cases, including 10 melanoma in situ cases.Expression of PITX1 was shown in nuclei of melanocytes in normal skin. PITX1 expression was positive (labeling index: 10%) in 21 (52.5%) cases and negative (labeling index: <10%) in 19 (47.5%) of 40 cases of primary cutaneous malignant melanoma. The mean tumor thickness in PITX1-negative cases (7.1110.3 mm) was significantly higher than that in the positive cases (1.903.19 mm) (P<0.01). The numbers of cases showing metastasis were 1 (4.76%) of 21 cases in PITX1-positive cases and 7 (36.8%) of 19 cases in PITX1-negative cases; the frequency was significantly higher in PITX1-negative cases than the positive cases (P=0.012). Moreover, the reduction in PITX1 expression correlated significantly with clinical stage (P<0.001). Interestingly, PITX1 expression was inversely correlated with cell proliferation of cutaneous malignant melanoma (P<0.001).Down-regulation of PITX1 expression might contribute to the progression of cutaneous malignant melanoma via promoting cell proliferative activity.


PubMed | Institute of Regenerative Medicine and Biofunction and Tottori University
Type: Journal Article | Journal: Journal of biochemistry | Year: 2016

Cu, Zn-superoxide dismutase (SOD1), an enzyme implicated in the progression of familial amyotrophic lateral sclerosis (fALS), forms amyloid fibrils under certain experimental conditions. As part of our efforts to understand ALS pathogenesis, in this study we found that reduction of the intramolecular disulfide bond destabilized the tertiary structure of metal free wild-type SOD1 and greatly enhanced fibril formation in vitro. We also identified fibril core peptides that are resistant to protease digestion by using mass spectroscopy and Edman degradation analyses. Three regions dispersed throughout the sequence were detected as fibril core sequences of SOD1. Interestingly, by using three synthetic peptides that correspond to these identified regions, we determined that each region was capable of fibril formation, either alone or in a mixture containing multiple peptides. It was also revealed that by reducing the disulfide bond and causing a decrease in the structural stability, the amyloid fibril formation of a familial mutant SOD1 G93A was accelerated even under physiological conditions. These results demonstrate that by destabilizing the structure of SOD1 by removing metal ions and breaking the intramolecular disulfide bridge, multiple fibril-forming core regions are exposed, which then interact with each another and form amyloid fibrils under physiological conditions.

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